Dietary benefits of fungal associates to an eruptive herbivore: potential implications of multiple associates on host population dynamics

We used the mountain pine beetle (Dendroctonus ponderosae Hopkins) and its two fungal associates, Grosmannia clavigera and Ophiostoma montium, to study potential nutritional benefits of fungi to bark beetles. We tested for potential effects of feeding on phloem colonized by fungi on beetle performance in field and laboratory studies. The fungi increased nitrogen levels in the phloem of attacked trees by 40%, indicating that it may be an important source of dietary nitrogen for mountain pine beetles. However, nitrogen levels of phloem inoculated with fungi in the laboratory were similar to uncolonized phloem, indicating that the fungi may redistribute nitrogen from the sapwood to the phloem rather than increase absolute levels of nitrogen. Beetles emerging from attacked trees carrying G. clavigera were larger than beetles carrying O. montium, which in turn were larger than beetles lacking fungi. Results of experimental laboratory studies varied, likely because of differences in the growth and sporulation of fungi under artificial conditions. Results indicate that the two fungi may offer complementary benefits to the mountain pine beetle because larvae preferentially fed on phloem colonized by both fungi together over phloem colonized by one fungus or uncolonized phloem. Teneral adults preemergence fed on spores in pupal chambers when they were produced and consumed little phloem before emerging. Teneral adults mined extensively in the phloem before emerging when spores were not produced in the pupal chamber. Our results provide evidence for a nutritional role of fungi in the diet of bark beetles and show that multiple associates may differentially affect beetle performance, which could have important implications for bark beetle population dynamics.     

Temporal variation in mycophagy and prevalence of fungi associated with developmental stages of Dendroctonus ponderosae (Coleoptera: Curculionidae)

Mycophagy by bark beetles is widespread. However, little is known regarding which developmental stages of bark beetles actually feed on fungi. To study this question, we sampled fungi associated with Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae) throughout development in naturally attacked trees. Isolations of fungi were made from phloem adjacent to brood and from brood exoskeletons and guts. Overall, the incidence of fungi with individual brood increased as brood development progressed. Grosmannia clavigera (Robinson-Jeffrey and Davidson) Zipfel, de Beer and Wingf. and Ophiostoma montium (Rumbold) von Arx exhibited generally opposing trends in prevalence. G. clavigera was most likely to be found in phloem adjacent to prewintering third- and postwintering fourth-instar larvae. O. montium was most likely to be found in phloem adjacent to eggs, first-instar larvae, pupae, and teneral adults. In contrast to isolations made from phloem, fungi isolated from brood guts and exoskeletons were not observed to shift in prevalence. First- and third-instar larvae were often observed migrating to older portions of their galleries, indicating that they do not spend all of their time feeding at, and extending, the apex of the gallery. Our results suggest that not only are D. ponderosae brood in contact with and feeding on fungi throughout development, but also, that during development, contact of brood with a particular fungus is likely to change. Such temporal shifts in fungal symbionts may be environmentally driven and have important implications in how these fungi interact with their hosts within and across generations.     

Target-specific PCR primers can detect and differentiate ophiostomatoid fungi from microbial communities associated with the mountain pine beetle Dendroctonus ponderosae

The aim of this study was to develop DNA probes that could identify the major fungal species associated with mountain pine beetles (MPB). The beetles are closely associated with fungal species that include ophiostomatoid fungi that can be difficult to differentiate morphologically. The most frequently isolated associates are the pine pathogens Grosmannia clavigera and Leptographium longiclavatum, the less pathogenic Ophiostoma montium, and an undescribed Ceratocystiopsis species (Cop. sp.). Because growing, isolating and extracting DNA from fungi vectored by MPB can be time and labour intensive, we designed three rDNA primer sets that specifically amplify short rDNA amplicons from O. montium, Cop. sp. and the pine Leptographium clade. We also designed two primer sets on a gene of unknown function that can differentiate G. clavigera and L. longiclavatum. We tested the primers on 76 fungal isolates that included MPB associates. The primers reliably identified their targets from DNA obtained from pure fungal cultures, pulverized beetles, beetle galleries, and tree phloem inoculated with G. clavigera. The primers will facilitate large-scale work on the ecology of the MPB-fungal-lodgepole pine ecosystem, as well as phytosanitary/quarantine sample screening.     

The impact of phloem nutrients on overwintering mountain pine beetles and their fungal symbionts

In the low nutrient environment of conifer bark, subcortical beetles often carry symbiotic fungi that concentrate nutrients in host tissues. Although bark beetles are known to benefit from these symbioses, whether this is because they survive better in nutrient-rich phloem is unknown. After manipulating phloem nutrition by fertilizing lodgepole pine trees (Pinus contorta Douglas var. latifolia), we found bolts from fertilized trees to contain more living individuals, and especially more pupae and teneral adults than bolts from unfertilized trees at our southern site. At our northern site, we found that a larger proportion of mountain pine beetle (Dendroctonus ponderosae Hopkins) larvae built pupal chambers in bolts from fertilized trees than in bolts from unfertilized trees. The symbiotic fungi of the mountain pine beetle also responded to fertilization. Two mutualistic fungi of bark beetles, Grosmannia clavigera (Rob.-Jeffr. & R. W. Davidson) Zipfel, Z. W. de Beer, & M. J. Wingf. and Leptographium longiclavatum Lee, S., J. J. Kim, & C. Breuil, doubled the nitrogen concentrations near the point of infection in the phloem of fertilized trees. These fungi were less capable of concentrating nitrogen in unfertilized trees. Thus, the fungal symbionts of mountain pine beetle enhance phloem nutrition and likely mediate the beneficial effects of fertilization on the survival and development of mountain pine beetle larvae.     

Survey and phylogenetic analysis of culturable microbes in the oral secretions of three bark beetle species

In a recent study, we reported a previously undescribed behavior in which a bark beetle exuded oral secretions containing bacteria that have antifungal properties, and hence defend their galleries against pervasive antagonistic Hyphomycete fungi. Actinobacteria, a group known for their antibiotic properties, were the most effective against fungi that invade the spruce beetle galleries. In the present study, we describe the isolation and identification of microorganisms from oral secretions of three bark beetles (Coleoptera: Curculionidae: Scolytinae): the spruce beetle, Dendroctonus rufipennis Kirby, the mountain pine beetle, Dendroctonus ponderosae Hopkins, and the pine engraver, Ips pini Say. Bacteria isolated from these three species span the major bacterial classes α-, β-, and γ-Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria, except for D. ponderosae , which yielded no α-proteobacteria or Bacteroidetes isolates. Spruce beetles and pine engraver beetles had similar numbers of α-proteobacteria isolates, but pine engravers yielded twice as many Bacteroidetes isolates as spruce beetles. In contrast, mountain pine beetles yielded more isolates in the β- and γ-proteobacteria than spruce beetles and pine engravers. The highest percentage of Actinobacteria was obtained from spruce beetles, followed by pine engravers and mountain pine beetles. All of the fungal isolates obtained from the three beetle species were Ascomycetes. The greatest fungal diversity was obtained in spruce beetles, which had nine species, followed by pine engravers with five, and mountain pine beetles with one.     

Competition and Coexistence In a Multi-partner Mutualism: Interactions Between two Fungal Symbionts of the Mountain Pine Beetle In Beetle-attacked Trees

Despite overlap in niches, two fungal symbionts of the mountain pine beetle (Dendroctonus ponderosae), Grosmannia clavigera and Ophiostoma montium, appear to coexist with one another and their bark beetle host in the phloem of trees. We sampled the percent of phloem colonized by fungi four times over 1 year to investigate the nature of the interaction between these two fungi and to determine how changing conditions in the tree (e.g., moisture) affect the interaction. Both fungi colonized phloem at similar rates; however, G. clavigera colonized a disproportionately larger amount of phloem than O. montium considering their relative prevalence in the beetle population. High phloem moisture appeared to inhibit fungal growth shortly after beetle attack; however, by 1 year, low phloem moisture likely inhibited fungal growth and survival. There was no inverse relationship between the percent of phloem colonized by G. clavigera only and O. montium only, which would indicate competition between the species. However, the percent of phloem colonized by G. clavigera and O. montium together decreased after 1 year, while the percent of phloem from which no fungi were isolated increased. A reduction in living fungi in the phloem at this time may have significant impacts on both beetles and fungi. These results indicate that exploitation competition occurred after a year when the two fungi colonized the phloem together, but we found no evidence of strong interference competition. Each species also maintained an exclusive area, which may promote coexistence of species with similar resource use.     

Fungus Symbionts Colonizing the Galleries of the Ambrosia Beetle <Emphasis Type="Italic">Platypus quercivorus</Emphasis>

Isolations were made to determine the fungal symbionts colonizing Platypus quercivorus beetle galleries of dead or dying Quercus laurifolia, Castanopsis cuspidata, Quercus serrata, Quercus crispula, and Quercus robur. For these studies, logs from oak wilt-killed trees were collected from Kyoto Prefecture, Japan. Fungi were isolated from the: (1) entrances of beetle galleries, (2) vertical galleries, (3) lateral galleries, and (4) the larval cradle of P. quercivorus in each host tree. Among the fungus colonies which appeared on YM agar plates, 1,219 were isolated as the representative isolates for fungus species inhabiting in the galleries based on their cultural characteristics. The validity of the visual classification of the fungus colonies was checked and if necessary properly corrected using microsatellite-primed PCR fingerprints. The nucleotide sequence of the D1/D2 region of the large subunit nuclear rRNA gene detected 38 fungus species (104 strains) of which three species, i.e., Candida sp. 3, Candida kashinagacola (both yeasts), and the filamentous fungus Raffaelea quercivora were isolated from all the tree species. The two yeasts were most prevalent in the interior of galleries, regardless of host tree species, suggesting their close association with the beetle. A culture-independent method, terminal restriction fragment length polymorphism (T-RFLP) analysis was also used to characterize the fungus flora of beetle galleries. T-RFLP patterns showed that yeast species belonging to the genus Ambrosiozyma frequently occurred on the gallery walls along with the two Candida species. Ours is the first report showing the specific fungi inhabiting the galleries of a platypodid ambrosia beetle.     

Mutual interactions between an invasive bark beetle and its associated fungi

Interactions between invasive insects and their fungal associates have important effects on the behavior, reproductive success, population dynamics and evolution of the organisms involved. The red turpentine beetle (RTB), Dendroctonus valens LeConte (Coleoptera: Scolytinae), an invasive forest pest in China, is closely associated with fungi. By carrying fungi on specialized structures in the exoskeleton, RTB inoculates fungi in the phloem of pines (when females dig galleries for egg laying and when males join them for mating). After eggs hatch, larvae gregariously feed on the phloem colonized by the fungi. We examined the effects of five isolates of RTB associated fungi (two from North America, Leptographium terebrantis and L. procerum, and three from China, Ophiostoma minus, L. sinoprocerum and L. procerum) on larval feeding activity, development and mortality. We also studied the effects of volatile chemicals produced in the beetle hindgut on fungal growth. Ophiostoma minus impaired feeding activity and reduced weight in RTB larvae. Leptographium sinoprocerum, L. terebrantis and L. procerum did not dramatically influence larval feeding and development compared to fungi-free controls. Larval mortality was not influenced by any of the tested fungi. Hindgut volatiles of RTB larvae, verbenol, myrtenol and myrtenal, inhibited growth rate of all the fungi. Our results not only show that D. valens associated fungus, O. minus, can be detrimental to its larvae; but, most importantly, they also show that these notorious beetles have an outstanding adaptive response evidenced by the ability to produce volatiles that inhibit growth of harmful fungus.     

黑龙江省长白落叶松八齿小蠹虫体及坑道的真菌类群

采集落叶松八齿小蠹成虫、幼虫、蛹,以及母坑道、子坑道、蛹室的韧皮组织和蓝变组织。采用马丁氏培养基和PDA综合培养基对各虫态体表、体内、坑道内真菌进行分离培养,统计真菌检出率。对获得的真菌菌株通过形态学和分子生物学相结合的方法进行鉴定。在落叶松八齿小蠹虫体内外和坑道内共分离出真菌24种,其中子囊菌7种,担子菌2种,无性型真菌15种。从各虫态体表共分离出真菌21种,从体内共分离出真菌6种,24种真菌在坑道内均有检出。其中,Ceratocystis fujiensis为检出率较高的蓝变真菌。     

合欢吉丁Agrilus subrobustus Saunders的学名厘定及危害特性

【目的】本文对危害合欢Albizia julibrissin Durazz的钻蛀性害虫合欢吉丁Agrilus subrobustus Saunders各虫态形态特征及危害特性进行了研究与描述,深入探索其虫道三维结构,以期为合欢吉丁的发生监测及防治提供依据。【方法】查阅相关文献,记述整理了合欢吉丁学名的变化过程,详细地观察并描述了剖腹卵、幼虫、蛹、成虫的形态特征;首次研究总结其坑道系统的各指标特点:虫口密度、坑道长宽、侵入孔特征、蛹室特征、羽化孔特征等,明确了完整虫道的发生发展规律;基于坑道拍照、各向量指标测量,对坑道进行科学手绘,使用Autodesk Maya软件完成合欢吉丁完整虫道的三维重建。【结果】合欢吉丁幼虫细长、乳白色,蛹为裸蛹。成虫铜绿色具金属光泽,观察对比其前胸背板长宽比、盘区和肩前隆脊形态,前胸腹板突形状,鞘翅柔毛分布位置,阳茎形态等特征,结合文献,明确其学名为Agrilus subrobustus Saunders。合欢吉丁主要危害树干韧皮部及木质部,具向阳性;在树皮下钻蛀坑道,侵入孔圆形,不超过1 mm;蛹室米粒状,多位于木质部,少数位于韧皮部;羽化孔"D"型;坑道形状具有规律性,可分为3种:"Z"字型及其变型,密集上下迂回型,椭圆或近半圆型坑道。【结论】基于形态特征的详细描述、学名厘定,有利于合欢吉丁的鉴定及学名的正确使用;完整虫道的复原便于在非成虫期结合寄主种类鉴定害虫种类、评估虫口密度和危害程度,为合欢吉丁的监测提供了具体的判别方法,为化学防治、生物防治等提供参考与技术支持。     

Reciprocal interactions between the bark beetle-associated yeast Ogataea pini and host plant phytochemistry

Here we report the first experiments testing reciprocal effects between the bark beetle-associated yeast, Ogataea pini, and phytochemicals present in tree tissues (Pinus ponderosa). We tested two hypotheses: (i) tree phytochemicals mediate O. pini growth and (ii) O. pini affects chemical composition of plant tissues. We tested six monoterpenes on O. pini biomass growth in vitro and found that most monoterpenes inhibited O. pini growth; however mean O. pini biomass increased 21.5% when treated with myrcene and 75.5% when treated with terpinolene, relative to control. Ogataea pini was grown on phloem tissue ex vivo to determine whether O. pini affected phloem chemistry. Monoterpene concentrations declined in phloem over time, but phloem colonized by O. pini had significantly different concentrations of monoterpenes at two periods than phloem with no yeast. After 7 d, when O. pini was present, concentrations of the monoterpene Δ-3-carene was 42.9% lower than uncolonized phloem and concentrations of the monoterpene terpinolene was 345.0% higher than uncolonized phloem. After 15 d phloem colonized by O. pini had 505.4% higher concentrations of α-pinene than uncolonized phloem. These experiments suggest that O. pini responds to phytochemicals present in host tissues and the presence of O. pini might alter the chemical environment of phloem tissues during the early stages of beetle development. The interactions between O. pini and phytochemicals in pine vascular tissues might have consequences for the bark beetle that vectors O. pini, Dendroctonus brevicomis.     

落叶松八齿小蠹体内外和坑道内真菌类群

采集落叶松八齿小蠹成虫、幼虫、蛹,以及母坑道、子坑道、蛹室的韧皮组织和蓝变组织。采用马丁氏培养基和PDA综合培养基对各虫态体表、体内、坑道内真菌进行分离培养,统计真菌检出率。对获得的真菌菌株通过形态学和分子生物学相结合的方法进行鉴定。在落叶松八齿小蠹虫体内外和坑道内共分离出真菌31种,其中接合菌3种,子囊菌8种,担子菌1种,半知菌19种。自各虫态体表共分离出真菌23种,体内共分离出真菌8种,31种真菌在坑道内均有检出。其中,Ceratocystis fujiensis为中国新记录种,Ophiostoma     

The Relative Abundance of Mountain Pine Beetle Fungal Associates Through the Beetle Life Cycle in Pine Trees

The mountain pine beetle (MPB) is a native bark beetle of western North America that attacks pine tree species, particularly lodgepole pine. It is closely associated with the ophiostomatoid ascomycetes Grosmannia clavigera, Leptographium longiclavatum, Ophiostoma montium, and Ceratocystiopsis sp.1, with which it is symbiotically associated. To develop a better understanding of interactions between beetles, fungi, and host trees, we used target-specific DNA primers with qPCR to assess the changes in fungal associate abundance over the stages of the MPB life cycle that occur in galleries under the bark of pine trees. Multivariate analysis of covariance identified statistically significant changes in the relative abundance of the fungi over the life cycle of the MPB. Univariate analysis of covariance identified a statistically significant increase in the abundance of Ceratocystiopsis sp.1 through the beetle life cycle, and pair-wise analysis showed that this increase occurs after the larval stage. In contrast, the abundance of O. montium and Leptographium species (G. clavigera, L. longiclavatum) did not change significantly through the MPB life cycle. From these results, the only fungus showing a significant increase in relative abundance has not been formally described and has been largely ignored by other MPB studies. Although our results were from only one site, in previous studies we have shown that the fungi described were all present in at least ten sites in British Columbia. We suggest that the role of Ceratocystiopsis sp.1 in the MPB system should be explored, particularly its potential as a source of nutrients for teneral adults.     

Laboratory study of conspecific avoidance by host-colonizingDendroctonus frontalis Zimm. (Coleoptera: Scolytidae)

Bark/phloem disk sandwiches (100 cm²) were colonized with one to six mating pairs of the southern pine beetle, Dendroctonus frontalis Zimm., to observe gallery construction and evaluate if avoidance behavior occurs between beetles. Frequencies of turn aways, touches, and crosses (three characteristics used to describe conspecific interaction) all increased significantly with beetle density. A threshold of three mating pairs within the 100- cm² phloem area was the density at which avoidance behavior became evident. At a threshold of five mating pairs the number of gallery crossings was significantly greater than at lower densities. Single and paired female beetles introduced into artificial galleries crossed empty neighboring galleries significantly more often than when neighboring galleries were occupied with other D. frontalis. The data suggest that D. frontalis females can regulate gallery spacing within the host through their ability to detect nearby beetles and their galleries. Possible spacing mechanisms are discussed.     

Distinguishing Ophiostoma ips and Ophiostoma montium,two bark beetle-associated sapstain fungi

Two synonymous sapstain species, Ophiostoma montium and Ophiostoma ips, which are vectored by Dendroctonus ponderosae and various bark beetles, respectively, were differentiated into separate species using growth and molecular characteristics. Analysis of 32 isolates of the two species from different countries showed that O. ips was able to grow at 35 degrees C while O. montium was not. This growth-based differentiation was strongly supported by sequence data for the internal transcribed spacer (ITS), 5.8S and partial 28S rDNA, and the beta-tubulin genes. The beta-tubulin gene sequence data indicate that the two species can easily be differentiated with a single polymerase chain reaction (PCR) assay.     

Saccharide-mediated antagonistic effects of bark beetle fungal associates on larvae

Bark beetles are among the most destructive of pine forest pests and they form close symbiotic relationships with ophiostomatoid fungi. Although some fungi are considered to be mutualistic symbionts of bark beetles with respect to the supply of nutrients, detrimental effects of fungal symbionts on larval growth have also been frequently reported. The mechanisms of such antagonistic effects are hypothesized to be a decrease in nutritional resources caused by competition for saccharides by the fungi. Here, we provide experimental evidence that three beetle-associated fungi modify the nutritional content of an artificial phloem diet, leading to a detrimental effect on the growth of Dendroctonus valens larvae. When larvae were fed a diet of pine phloem in agar medium colonized with any of these fungi, feeding activity was not affected but weight significantly decreased. Additional analysis showed that fungi depleted the fructose and glucose concentrations in the phloem media. Furthermore, these detrimental effects were neutralized by supplementing the media with fructose or glucose, suggesting that fungi may affect larval growth by modifying diet saccharide contents. These data indicate that fungus-induced nutritional changes in bark beetle diet can affect larval growth, and that the mechanism involves fungus-induced saccharide depletion from the larval diet.     

Bacteria in oral secretions of an endophytic insect inhibit antagonistic fungi

Abstract.  1. Colonisation of host trees by an endophytic herbivore, the spruce beetle, Dendroctonus rufipennis , is accompanied by invasion of its galleries by a number of fungal species. Four of these associated species were identified as Leptographium abietinum , Aspergillus fumigatus , Aspergillus nomius , and Trichoderma harzianum .
2.  Trichoderma and Aspergillus significantly reduced spruce beetle survival and reproduction in controlled assays.
3. A previously undescribed behaviour was observed, in which spruce beetle adults exuded oral secretions, especially within fungus-pervaded galleries.
4. These oral secretions inhibited the growth of fungi except A. nomius , and disrupted the morphology of the latter. Administration of these secretions indicated a dose-dependent inhibitory effect.
5. Oral secretions cultured on microbiological media yielded substantial bacterial growth.
6. Filter-sterilised secretions failed to inhibit fungal growth, evidence that the bacteria are responsible for the antifungal activity.
7. Nine bacterial isolates belonging to the Actinobacteria, Firmicutes, Gammaproteobacteria, and Betaproteobacteria taxa were obtained from the secretions.
8. Bacterial isolates showed species-specific inhibitory activity against the four fungi antagonistic to spruce beetle. The bacterium with the strongest fungal inhibition activity was the actinomycete Micrococcus luteus .
9. The production of bark beetle secretions containing bacteria that inhibit fungal growth is a novel finding. This suggests an additional level of complexity to ecological associations among bark beetles, conifers, and microorganisms, and an important adaptation for colonising subcortical tissue.     

Symbiotic interactions of culturable microbes with the nickel hyperaccumulator Berkheya coddii and the herbivorous insect Chrysolina clathrata

Chrysolina clathrata is a specialized phytophagous beetle feeding exclusively on the herbaceous nickel hyperaccumulating plant Berkheya coddii. These organisms appear impervious to the toxic levels of nickel in their environment. In the current study we aimed to identify microorganisms that may have symbiotic relationships with these organisms. Culture techniques were used to isolate bacteria and fungi from plants and the faeces of beetles reared under laboratory conditions. The identity of isolates was determined using morphology and molecular techniques. Several genera of filamentous fungi (Alternaria, Aspergillus, Bipolaris, Cladosporium, Epicoccum, Fusarium, and Penicillium), yeasts (Cryptococcus, Meyerozyma, and Rhodotorula), as well as endophytic bacteria (Bacillus and Lysinibacillus) were isolated from the leaves of the plant. We also selectively isolated yeasts (Candida, Cryptococcus, Debaryomyces, Meyerozyma and Wickerhamomyces) from the beetle’s faeces. Subsequently we determined the minimum inhibitory Ni-concentration (MIC) of all isolates. The endophytic bacteria, filamentous fungi and the yeasts Candida intermedia, Cryptococcus flavescens and Meyerozyma guilliermondii, showed notable Ni resistance, while the beetle’s gut seems to select for Ni resistant yeasts.     

Colorado potato beetle manipulates plant defenses in local and systemic leaves

Herbivore microbial associates can affect diverse interactions between plants and insect herbivores. Some insect symbionts enable herbivores to expand host plant range or to facilitate host plant use by modifying plant physiology. However, little attention has been paid to the role of herbivore-associated microbes in manipulating plant defenses. We have recently shown that Colorado potato beetle secrete the symbiotic bacteria to suppress plant defenses. The bacteria in oral secretions from the beetle hijack defense signaling pathways of host plants and the suppression of induced plant defenses benefits the beetle’s performance. While the defense suppression by the beetle-associated bacteria has been investigated in local damaged leaves, little is known about the effects of the symbiotic bacteria on the manipulation of plant defenses in systemic undamaged leaves. Here, we demonstrate that the symbiotic bacteria suppress plant defenses in both local and systemic tissues when plants are attacked by antibiotic-untreated larvae.